JPH02203794A - Monoclonal antibody for oncogene product - Google Patents

Abstract

PURPOSE:To obtain a monoclonal antibody which specifically reacts with a protein corresponding to cellular oncogene, c-myb, to enable higher accurate diagnosis of the canceration of hematopoietic cells. CONSTITUTION:The immunoassay of the protein corresponding to cellular oncogene, c-myb, is effected using a combination of a monoclonal antibody which reacts with the whole protein of about 85 kilodalton, corresponding to a cellular oncogene and also reacts with the protein which is formed by removing the peptide of 35 kilodalton from the C-terminal side of the whole protein, and another monoclonal antibody which reacts with the whole protein of about 85 kilodalton, corresponding to cellular oncogene, c-myb, but does not reacts with the protein which is formed by removing the peptide of about 35 kilodalton from the C-terminal side of the whole protein.

Description

DETAILED DESCRIPTION OF THE INVENTION [Industrial Application Field] This invention relates to a novel monoclonal antibody, a hybridoma cell secreting the monoclonal antibody, and an immunoassay method using the monoclonal antibody.

[Conventional technology]

A method for diagnosing cancer by measuring the amount of oncogene products, ie, proteins corresponding to oncogenes, is known. As a method for diagnosing canceration of hematopoietic cells, cellular oncogene c-
A method for measuring the concentration of a protein corresponding to myb (hereinafter sometimes referred to as c-myb protein) in cell lysate has been known.

[Problem to be solved by the invention]

However, in conventional immunoassays, c-my
Since polyclonal antibodies contained in antisera obtained from animals immunized with c-myb protein were used, they had the disadvantage of low specificity (lack of reproducibility). We provide protein-specific monoclonal antibodies and highly specific and reproducible c-
An object of the present invention is to provide an immunoassay method for myb protein.

[Means to solve the problem]

The above object, according to the present invention, (1) reacts with the entire protein of about 85 kilodaltons (hereinafter sometimes abbreviated as whole c-myb protein) corresponding to the cellular oncogene c-myb; A monoclonal antibody that also reacts with a protein in which about 35 kilodaltons of the C-terminal peptide have been removed from the entire protein of about 85 kilodaltons (i.e., the whole c-+++yb protein) (hereinafter sometimes abbreviated as c-myb protein 50); and (2) reacts with all c-myb proteins, but c-myb
This can be achieved using a monoclonal antibody that does not react with protein 50.

Furthermore, the present invention provides (a) cells formed by fusion of mouse spleen cells and mouse myeloma cells immunized with whole c-ayb protein;
and (b) reacts with all c-myb proteins, and c-a+y
The present invention relates to a hybridoma cell characterized in that it secretes a monoclonal antibody that also reacts with b protein 50.

Furthermore, the present invention provides: (a) formed by the fusion of mouse spleen cells and mouse myeloma cells immunized with whole c-myb protein;
and (b) reacts with all c-myb proteins, but c-myb
The present invention relates to a hybridoma cell characterized by secreting a monoclonal antibody that does not react with protein 50.

Furthermore, the present invention is characterized in that one or more monoclonal antibodies against a protein corresponding to the cellular oncogene c-myb are used to detect the cellular oncogene c-a+y in an aqueous liquid.
The present invention also relates to a method for immunoassaying the protein corresponding to b.

The present invention will be explained in detail below.

Anti-C-liny according to the present invention, which is a monoclonal antibody against the protein corresponding to the cellular oncogene c-myb gene
b protein monoclonal antibodies can be produced by culturing novel mouse hybridoma cells in vitro (eg, in culture) or in vivo (eg, intraperitoneally in mice).

The mouse hybridoma cells used here are: - Mouse spleen cells immunized with c-myb protein and mouse myeloma cells (myeloma cells) were used for boat fishing.
and MilsLein's method (Nature, No. 25)
6, p. 495 (1975)], it can be produced by cell fusion.

Further, as a medium for culturing the above-mentioned hybridoma cells, any medium suitable for culturing hybridoma cells can be used, and Dulbecco's modified Eea medium is preferably used.
gle's n+edium H (hereinafter referred to as DME)
A medium containing fetal bovine serum, L-glutamine, L-pyruvic acid, and antibiotics (penicillin G and streptomycin) is used.

The above hybridoma cells may be cultured in vitro at 5% C0zrj 1 degree and 37°C in a culture medium, for example.
The culture is carried out for about 3 days, or for about 14 days when cultured in vivo, for example, intraperitoneally in mice.

When separating and purifying monoclonal antibodies from the culture fluid or mouse ascites fluid thus produced, it is possible to use methods commonly used for protein isolation and purification. Such methods include ammonium sulfate salting out, ion exchange chromatography, molecular sieve column chromatography using molecular sieve gel, protein A bond length II
There are methods such as affinity column chromatography, dialysis, and freeze-drying.

The anti-c-syb protein monoclonal antibody thus obtained does not react with other oncogene products, and the anti-c-syb protein monoclonal antibody
It has the ability to specifically bind only to myb protein, and c-
It is useful as a reagent for immunoassay of myb protein.

Anti-C-Month yb protein monoclonal antibodies of this invention (for example, ?1YB-1, MYB-2 obtained in the Examples described below)
NYB-3 and MYB-4) can be used as reagents in various immunoassay methods.

For example, immunoplot quantification is generally carried out using one or more anti-C1 siB protein monoclonal antibodies. First, the analyte (e.g. cell lysate) is
treated by electrophoresis using DS-polyacrydamide,
Next, the cell lysate in this gel is transferred to a nitrocellulose membrane or other membrane, and the transferred membrane is coated with anti-c-Ilyb.
One or more protein monoclonal antibodies (for example, the monoclonal antibodies MYB-1 and/or the monoclonal antibodies of the present invention)
Or MYB-3) is immersed in a solution for about 1 hour, then washed, an enzyme-labeled anti-mouse γ-globulin antibody is added, and the sample is left to stand for about 1 hour. After washing, an enzyme substrate solution is added and an enzyme reaction is carried out for about 30 minutes. After the reaction is completed, the c-syb protein in the sample is quantified by measuring the products of the enzymatic reaction by a colorimetric method using a densitometer.

In addition, two types of monoclonal antibodies (c-
syb proteins), one in insoluble form (e.g. attached to the wells of a microtiter plate) and the other labeled (e.g. radioisotope, enzyme, luminescent). c- in the aqueous liquid sample (e.g. cell lysate).
It can be used in immunoassay based on binding with myb protein. For example, enzyme immunoassay is generally performed using two types of anti-c-myb protein monoclonal antibodies that recognize mutually different antigenic determinants of c-myb protein. First, a well (hole) of a microtiter plate or a plastic tube is filled with one type of antibody (for example, the monoclonal antibody MYB according to the present invention).
-1) to sensitize. Next, a solution of an analyte containing c-myb protein (e.g., cell lysate) and an enzyme-labeled antibody of another type (e.g., monoclonal antibody M'/B-3 according to the present invention) is placed in this well or plastic tube. After standing for 30 minutes, the plate is washed, an enzyme substrate solution is added, and an enzyme reaction is carried out for about 30 minutes. After the reaction is completed, the c-myb protein in the sample is quantified by colorimetry or the like.

In addition, chemiluminescence immunoassay method is based on -a, c-+my
This is carried out using two types of anti-c-myb protein monoclonal antibodies that recognize different antigenic determinants of b protein. First, a well of a microtiter plate or a plastic tube is sensitized with one type of antibody (for example, the monoclonal antibody MYB-1 of the present invention). Next, add c-+ayb to this well or plastic tube.
A solution of a protein-containing analyte (e.g., cell lysate) and another type of antibody labeled with a chemiluminescent compound (e.g., monoclonal antibody MYB-3 according to the present invention) is added, allowed to stand for about 30 minutes, then washed, and hydrogen peroxide is removed. In addition, chemiluminescence is caused, and this is detected using a luminometer to detect c-+a in the sample.
Quantify yb protein.

〔Example〕

Next, this invention will be explained in more detail with reference to examples.
This invention is not limited by the following examples.

1: Preparation of the c-myb protein of c-mb was described in Gene, 56 (19
87) It was carried out according to the method described in 125-135. That is, the expression vector pAR2156 containing the cDNA of the mouse oncogene c-myb was injected into Escherichia coli (1.5 her
E. coli BL21 (pAR2156) obtained by transforming the BL21 strain (Ichia colt)
) strain (RIKEN Scene Bank Cell Bank) was sufficiently grown in culture medium. Next, isopropyl-β, a non-metabolizable inducer of the lactose operon.

D-thiogalactoside (IPTG) was added to the culture medium.

After culturing for 1 hour, E. coli BL21 (pAR2156
) strain was collected, the bacterial lysate was electrophoresed on 5DS-polyacrylamide, and the gel containing the c-myb protein, identified by its molecular weight (85 kilodaltons), was cut out.

The c-myb protein was electrophoretically extracted from this gel. The c-myb protein thus obtained (A280nm=
0.2.

1O-) was used as an immunogen and as an antigen for HLISA to select anti-c-myb protein monoclonal antibody-producing bilidoma.

(a) Preparation of immunized spleen cells: The above c-a+yb protein immunogen solution (A280rv=
0.1) was mixed with an equal amount of Freund's complete adjuvant until emulsified, and the mixture 200I was intraperitoneally administered to mice (first immunization).
. After 30 days, the mouse was given 200 ml of the same mixture as above.
I was administered intraperitoneally to mice (second immunization). 21 days after the second immunization, c-11yb protein immunogen solution (
A280nm = 0.1) was diluted with an equal volume of physiological saline, and the diluted solution 200I was intravenously administered to the mouse (final immunization). Three days after the final immunization, the spleen was aseptically removed from the mouse and used for the next cell fusion step.

(b) Cell fusion: The spleen removed aseptically was placed in a Petri dish containing DME medium containing 15% fetal bovine serum.
After the spleen was perfused with approximately 15 ml of DME medium containing 15% fetal bovine serum to drain out the spleen cells, the spleen cell suspension was passed through a nylon mesh. The spleen cells were collected in a 50d centrifuge tube and centrifuged at 500Xg for 10 minutes. Hemolizing solution (155ai) was added to the pellet thus obtained.
MNH, CA'.

10mM KHCOz, 1mM Na1EDTA pH
7,0) 5 m was added and suspended. The suspension was left at 0° C. for 5 minutes to destroy red blood cells in the suspension. DME medium containing 15% fetal bovine serum 10 to 20 was added and centrifuged. The cell pellet thus obtained was washed with DME medium by centrifugation, and the number of living spleen cells was determined.

On the other hand, 1 x 10' of the above spleen cells were added to about 2 x 10' mouse myeloma cells (myeloma cells) SP 210-Ag 14 (RIKEN Scene Bank Cell Bank) that had been cultured in advance, and the mixture was placed in DME medium. The mixture was mixed well and centrifuged (500×g, 10 minutes). Aspirate the supernatant, loosen the pellet well, and dissolve in 40% polyethylene glycol 4000 solution (kept at 38°C).
．． The polyethylene glycol solution and cell pellet were mixed by adding 5-ml dropwise and gently rotating the centrifuge tube by hand for 1 minute. Next, the DME medium kept at 38°C was mixed with 1-ml solution every 30 seconds. In addition, the tube was rotated gently.

After repeating this operation 10 times, 20% 15% fetal bovine serum
Add DME medium containing ~30w11 and centrifuge (5
00x g for 10 minutes). After removing the supernatant, the cell pellet was mixed with HT medium (DME medium containing 15% fetal bovine serum) containing aminopterin 4X10-'M1 thymidine 1.6X10-'M, hypoxanthine lx10-'
' M), and then centrifuged to obtain 2
After washing twice, the cells were suspended in 40-ml of the above HT medium. Add 20 cells of this cell suspension to each well of a 96-well cell culture plate.
0 pZ was dispensed and culture was started at 37° C. in a carbon dioxide gas incubator containing 5% carbon dioxide gas. During culturing, approximately 100 m of the medium in each well was removed at intervals of 2 to 3 days, and 1 ml of the above-mentioned HT medium was newly added to select hybridomas that proliferated in the HT medium. From day 8, HT medium containing 15% fetal bovine serum (1.6 x 1 thymidine in DME medium)
The mixture was replaced with 0-5M1hyboxanthin lX10-'M), the growth of the hybridomas was observed, and on the 100th day, hybridomas producing anti-c-myb protein antibodies were screened by the above-mentioned ELISA method.

(C) Establishment of hybridoma The presence or absence of produced antibodies in the hybridoma culture supernatant is determined by ELIS.
Measured by method A. 96 well I! LISA play) (1-■ulonll, Japan Guinatec Co., Ltd.)
Add the purified c-myb protein solution (A2
80rv = 0.05, diluted with saline) 51J
After dispensing 0.0
After washing three times with 5% Tween 20-saline,
50 pl of culture medium was added to each well and reacted at 25°C for 1 hour.

Next, 50Ii of peroxidase-conjugated anti-mouse antibody (Dako, Denmark) diluted 200-fold in Tween 20-saline was added to each well. After the reaction is completed, 0.
Wash each well three times with 0.05% Tween 20 - saline solution 2 containing 0.5 mM aminoantipyrine, 10 mM phenol and o, oos% hydrogen peroxide.
50111 was added to each well, reacted at 25°C for 30 minutes, and the absorbance at 490n* of each well was measured.

As a result, antibody production was observed in 4 out of 192 wells. Each hybridoma in the 4 wells was transferred to a 24-well plate and cultured for 4 to 5 days in HT medium containing 15% fetal bovine serum. Thereafter, the presence or absence of production of anti-c-myb protein antibodies was confirmed again by ELISA, and then cloning was performed by limiting dilution. The limiting dilution method is H
A cell suspension diluted with T medium to give 5 bilidomas/- was placed into each well of a 96-well plate containing 2 x 10' normal BALB/C mouse peritoneal cells per well. Dispensed. Ten days later, hybridoma clones producing anti-c-syb protein-specific antibodies were screened by ELISA. As a result, for each hybridoma, 20 to 4
0 antibody producing clones were obtained. Among these clones, clones with good proliferation, high antibody secretion ability, and stability were selected and re-cloned in the same manner as described above to obtain anti-ca+yb protein-specific antibody-producing hybridoma MYB-1. , MYB-2゜MYB-3 and MYB
-4 was established.

3: Monochrome (a) In vitro method mouse hybrid-7MVB-1, MYB-2, MYB
-3 and MYB-4 were each cultured in DME medium containing 15% fetal bovine serum at 37°C in a 5% carbon dioxide atmosphere for 72 to 96 hours. Centrifuge the culture (
After 10000 x g (10 min), solid ammonium sulfate was slowly added to the supernatant to a final concentration of 50%. The mixture was stirred under water cooling for 30 min, then left for 60 min, and after centrifugation (10000 x g, 10 min),
The obtained precipitate was added to a small amount of 10i+M phosphate buffer (pH 8
, O) and dialyzed against 1000 times the volume of 10s+M phosphate buffer. This was loaded onto a column of DEAR-cellulose already equilibrated with 10+sM phosphate buffer. Elution of monoclonal antibodies was carried out using loa+M phosphate buffer (pH 8,0) and 0.2 M Na (1 containing J
The concentration gradient method was performed between 0 mM phosphate buffer (pH 18,0). The eluted monoclonal antibody was concentrated by ultrafiltration and added to 0.1M phosphate buffer (pH 8.0).
Dialyzed against. To remove 1 g of bovine serum, the dialysate was passed through a column of 1 g of goat serum - Sepharose 4B. The flow-through was then diluted with 0.1 M phosphate buffer (pH
Protein A-Sepharose 4B equilibrated with 8,0)
was packed into a column. The column was eluted with a pH 3.5 buffer to remove the purified anti-c-myb protein specific antibody MYB-
1 (Similarly, MYB-2, MYB-3 and MYB-
A solution of 4) was obtained.

(b) In vivo method 0.5 mj of pristane (2,6,10,14-tetramethylpentadecane) was administered intraperitoneally to 10-12 week old BALB/C mice 14-20 days after the intraperitoneal injection. Hybridomas MYB-1, MYB-2, MYB-3, or MYII-4 grown in vitro were injected into mice.
The cells were inoculated at 2 x 106 cells per animal.

Approximately 10-15 from one mouse per hybridoma
Ascites of m was obtained. The antibody concentration is 2-10qr/
It was affi. The monoclonal antibody in ascites was purified in the same manner as the in vitro purification method described above (with the exception of passing the antibody through a 1gG-Sepharose 4B column of goat anti-sugar serum).

Country A4: Monochrome globulin anti-c-
ayb protein-specific monoclonal antibody MYB-1゜MY
Identification of the immunoglobulin class and specificity of B-2, MYII-3 and elbow B-4 was performed by offtelony immunodiffusion and enzyme immunoassay, respectively. The results are shown in Table 1.

Table 1 Various amounts of c-myb protein were produced in the following cells, and the amount of c-+wyb protein in the cell lysate of each cell was quantified by immunoplotting. The cells used are as follows. 5tlP? -1 (T-NIIL) (RIKEN Scene Bank Cell Bank), RPM-1 (T-NIIL)
-ALL) [RIKEN Scene Bank Cell Bank], M
OLT4 (T-ALL) (RIKEN Scene Bank Cell Bank), KE-37 (RIKEN Scene Bank Cell Bank), T-1 (RIKEN Scene Bank Cell Bank), RB22Pre T/B v-abl trait Transformed cell line (expressing all c-ayb proteins) [RIKEN Scene Bank Cell Bank], Ts* c-myb
Transformed cell line (expressing c-+ayb protein 50) [RIKEN Scene Bank Cell Bank], 37
3-line fibroblast cell line (c-myb protein is not expressed) (RIKEN Scene Bank Cell Bank). Each cell (5X10"cells/me) was treated with 50mM Na
Cj! , 0.5% deoxycholic acid (deoxy
cholate), 0.5% NP-40 and 0.1
101 Tris-HCl buffer (pH 7.5) containing % SDS
) to lyse the cells (4°C). Centrifuge each cell lysate (10,0 OOX g.

(4°C) for 15 minutes, and the supernatant was collected. After each supernatant 20I11 was subjected to 5DS-polyacrylamide electrophoresis, the proteins in the gel were transferred to a nitrocellulose filter. This filter was mixed with 3% skim milk and 0.15M
Na (10a + M containing J) Lis-HCl buffer (pH 7
, 5) at 25°C for 1 hour, anti-c-+++yb protein monoclonal antibody CMVB-1 was used as the secondary antibody.
, MYB-2, MYB-3, MYB-4, and ascites fluid collected by administering SP 210 cells (as a blank) into the peritoneal cavity of a mouse) were allowed to react at 25° C. for 1 hour. After washing the filter 5 times with 10mM Tris-saline (pH 7.5), the secondary antibody alkaline phosphaptase-labeled anti-mouse IgG was added.
was reacted at 25° C. for 1 hour, and washed in the same manner as above. 1
．． In 50IIIM carbonate buffer (pH 9,8) containing 5 mM magnesium chloride, nitroblue tetrazolim at a final concentration of 0.33 μ/− and 5-bromo-4-chloro-3-indo at a final concentration of 0.17 μ/1 d. Ilphosfe) p)
A solution containing luidine was used as a coloring solution. This coloring solution was reacted at 25° C. for 5 to 10 minutes, and the reaction was stopped by washing the filter with water. The intensity of the band that appeared was quantified using a densitometer. The results (Western blotting method) are shown in FIG. In Figure 1, lane A is RB22 Pre T/Bv-ab
l transformed cell line (expressing the entire c-myb protein),
Lane B is the T@q CT myb transformed cell line (
c-myb protein 50 is expressed) and C lane is 3T3.
This is a fibroblast cell line (c-myb protein is not expressed).

As is clear from Figure 1, the monoclonal antibody MYB
-1 and MYB-2 are 85 kilodalton c-myb
It reacts with proteins, but does not react with c-myb protein 50. On the other hand, monoclonal antibodies MYB-3 and MY
B-4 is an 85 kilodalton c-myb protein and a c-m
Reacts with both yb protein 50. Therefore, monoclonal antibodies MYB-1 and MYB-2 are c-myb
The N-terminal region of the protein was isolated using the monoclonal antibody MYB.
-3 and MYB-4 recognize the C-terminal region.

FIG. 2 shows the amount of c-myb protein expressed in various cells by monoclonal antibody MVB-1.

The above cell line was treated in the same manner as in Example 4, immunoplot was performed, and the intensity of the c-a+yb protein band was measured with a densitometer and graphed.

Disaster 1 Ken: N.S.P. Santoi Chinakane and Kawaoi [Journal of Histochemistry]
and Cytochemistry (Journal of
Histochemistry and Cytoch
emistry) Vol. 22, pp. 1084-1091 (1
[974], horseradish peroxidase was mixed with anti-c-myb protein monoclonal antibody MVB.
-1.

Using this enzyme-labeled antibody, one of the c-myb proteins was isolated.
Step sandwich enzyme immunoassay was performed as follows.

100 μl of 20a+M carbonate buffer containing monoclonal antibody MYB-2 at a concentration of 10×/− was placed in a 96-well flat-bottomed polystyrene microtiter plate and allowed to stand at 25° C. for 30 minutes. 0.05 that plate
Washed three times with physiological saline containing % Tween-20.

Into the wells of the plate sensitized with antibodies in this way, 50 I
, and the peroxidase-labeled antibody prepared above and 0
．． 20 containing 15M Na(J and 2% bovine albumin)
-h 100I11 of phosphate buffer (pH 8,0) was added. After standing at 25°C for 30 minutes, the plate was diluted with 0.05% Tw.
Washed three times with physiological saline containing een-20. Next, 200111 enzyme substrate solutions (a solution containing 10 mM phenol, 2 (1+M4-aminoantipyrine, and 0.005% hydrogen peroxide)) were added to each well of the plate. After reacting at 25°C for 30 minutes, each well was liquid 4
The absorbance at 90 Mm was measured with an MP-590 Micro Elizer Mini Reader (manufactured by Dynatic). The amount of c-myb protein in the sample was determined from a calibration curve drawn from the absorbance at 490 Mm of the calibration c-myb protein measured at the same time, and was 10 Ig/- to 110
Good data could be obtained in the range of 0n/-.

:' As a method for binding a chemiluminescent substance to a monoclonal antibody, a chemiluminescent substance into which a succinimide group has been introduced into the shell is mixed with the antibody, and the unbound chemiluminescent substance is removed using a PD-10 column. (Pharmacia). In this example, the antibody is a monoclonal antibody) IVB
-1 and Acridiniu as a chemiluminescent substance.
+m-1 (Dojin) was used. Add 100ml of 20mM carbonate buffer containing monoclonal antibody MYB-2 at a concentration of 10n/1 to a plastic tube and incubate at 25°C.
It was left undisturbed for 1 hour.

The tube was washed three times with saline containing 0.05% Tween-20. Into the tube sensitized with the antibody in this way, the test substance (8M urea, 0.5% containing 1% SO3) was added.
Cell lysate treated with 1 M Tris buffer) 501
1 and Acridiniu+s-1 labeled monoclonal antibody MYB-1 prepared above and 0.15M NaC
100 μl of 20 mM phosphate buffer (pH 8, O) containing 1% and 2% bovine albumin was added. After standing at 25° C. for 1 hour, the tube was washed three times with physiological saline containing 0.05% Tween-20. Then 1011MHzO
Add i-liquid 2004 and adjust the luminescence amount to TD-4000L [IM
As a result of measurement with IPHOMETER (LABO5ctence), good data could be obtained in the range of 1 pg/d to 10 ng/yd.

〔Effect of the invention〕

The specific monoclonal antibody of this invention is c-+myb
An effective method for immunoassay of proteins can be provided.

[Brief explanation of the drawing]

Figure 1 shows c of four monoclonal antibodies according to the present invention.
FIG. 2 is a graph showing the expression level of c-myb protein in various cells by the monoclonal antibody according to the present invention.

Claims (1)

[Scope of Claims] 1. Reacts with the entire protein of approximately 85 kilodaltons corresponding to the cellular oncogene c-myb, and moreover, the C-terminal peptide of approximately 35 kilodaltons is removed from the entire protein of approximately 85 kilodaltons. A monoclonal antibody that also reacts with other proteins. 2. It reacts with the entire protein of about 85 kilodaltons corresponding to the cellular oncogene c-myb, but it reacts with the protein from which the C-terminal peptide of about 35 kilodaltons has been removed from the entire protein of about 85 kilodaltons. No monoclonal antibodies. 3, (a) formed by the fusion of mouse myeloma cells with spleen cells of a mouse immunized with a protein corresponding to the cellular oncogene c-myb, and (b) a protein corresponding to the cellular oncogene c-myb. Secreting a monoclonal antibody that reacts with the entire 85 kilodalton protein and also reacts with a protein in which about 35 kilodaltons of the C-terminal peptide have been removed from the entire about 85 kilodalton protein.
A hybridoma cell characterized by: 4, (a) formed by the fusion of mouse myeloma cells with spleen cells of a mouse immunized with a protein corresponding to the cellular oncogene c-myb, and (b) a protein corresponding to the cellular oncogene c-myb. A hybridoma characterized in that it secretes a monoclonal antibody that reacts with the entire 85-kilodalton protein but does not react with a protein in which about 35 kilodaltons of the C-terminal peptide have been removed from the entire about 85-kilodalton protein. cell. 5. A method for immunoassay of a protein corresponding to the cellular oncogene c-myb in an aqueous liquid, which comprises using one or more monoclonal antibodies against the protein corresponding to the cellular oncogene c-myb.